DESCRIPTION: Capsular polysaccharide of Staphylococcus aureus is a potential virulence factor responsible for the pathogenicity of the organism. The molecular biology of capsule production, however is as yet poorly understood. The long term goals of this project are to study the molecular control of staphylococcal capsule synthesis and its role in the pathogenesis of S. aureus. We have constructed a plasmid library of S. aureus strain M. Screening of the library has resulted in a clone containing a recombinant plasmid which complemented a negative capsular mutant derived from strain M. The nearby capsular genes (cap) have also been cloned by chromosome walking technique. Other genes not clustered together will be cloned from S. aureus strain M by screening the library for complementing a mutant which carries a mutation outside the cluster. The cloned cap genes will be subcloned and the subclones will be used to complement various mutations to determine the minimum number of genes. The gene products will be identified and correlated to their corresponding genes by maxicell or minicell expression of the cloned fragments. Promoters will be localized by Northern analysis or by cloning into a promoter-cloning vector. These experiments will allow the determination of the number of the operons and genes involved, and how the genes are transcribed and organized. A gene product will be correlated to its function in the capsular biosynthetic pathway by analyzing the phenotypic change resulted from the mutation of the gene. The location of the genes on the chromosome will be mapped by a combination of pulse-field gel electrophoresis and Southern analysis followed by transformation. Genetic and biochemical experiments will be carried out to study the molecular basis for the phenotypic loss of capsule production in strain M. The successful completion of the studies outlined in this proposal will significantly advance our understanding on the molecular genetic of capsule of S. aureus which will, in turn, provide a firm conceptual basis for studying the role of capsule in staphylococcal pathogenesis.